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Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021

DRC 8021 Biosafety Level-3

Laboratory

Facility Manual

Durham Research Center University of Nebraska Medical Center

Omaha, Nebraska 2006

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 Table of Contents: SECTION 1: Introduction 1.1 Purpose 1.2 Scope 1.3 Biosafety Training 1.4 Biosecurity Clearance 1.5 Employee Responsibility SECTION 2: Administration 2.1 Laboratory Director-SA 2.2 Safety and Security Manager 2.3 Facility Manager 2.4 Institutional Biosafety Officer 2.5 Laboratory Advisory Council Members SECTION 3: Administrative Duties 3.1 Laboratory Director-SA 3.2 Safety and Security Manager 3.3 Facility Manager 3.4 Institutional Biosafety Officer 3.5 Laboratory Advisory Council SECTION 4: Biosecurity and Biosafety Personnel Clearance Requirements 4.1 Procedure 4.2 Requirements for Working with Select Agents 4.3 Training Requirements 4.4 Final Approval 4.5 Personnel Changes 4.6 Annual Requirements

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 SECTION 5: Laboratory Biosecurity 5.1 Personnel Requirements for Laboratory Access 5.2 Environmental Services 5.3 Maintenance Personnel Access 5.4 Security Breaches and Emergencies SECTION 6: Personal Protective Equipment 6.1 Introduction 6.2 Responsibilities 6.3 Selection and Use of Personal Protective Equipment 6.4 Training 6.5 Recordkeeping SECTION 7: Laboratory Signage 7.1 Proper Use of Signs and Tags 7.2 Posting of Signs and Tags 7.3 Hazard Warning Signs and Labels 7.4 Examples of Signs SECTION 8: Workflow: Laboratory Entrance and Egress 8.1 Entering the Lab

8.2 Working at the Biological Safety Caninet (BSC) –Suite C,D &F 8.3 Maintenance and Cleaning

8.3 Exiting the Lab SECTION 9: Laboratory Equipment 9.1 General Notes 9.2 Equipment Certification and Safety Checks 9.3 Gas Canisters 9.4 Laboratory Exhaust System 9.5 Biological Safety Cabinets 9.6 Centrifuge Procedure 9.7 Ultra-Centrifuges 9.8 Ultra-low freezers

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 9.9 Equipment Decontamination 9.10 Biological Safety Cabinet Decontamination 9.11 Laboratory Facility Decontamination SECTION 10: Working in a Biological Safety Cabinet 10.1 Prior to Beginning Work 10.2 While working in the BSC 10.3 Upon completion of Work SECTION 11: Decontamination of Biohazardous Waste 11.1 Standard Procedures 11.2 Autoclave Performance Monitoring SECTION 12: Spill Clean-up Procedure 12.1 Spills of Biological Agents 12.2 Personal Protective Equipment 12.3 Eye and Skin Involvement 12.4 Chemical Spills SECTION 13: Emergency Procedures 13.1 Call-down Phones 13.2 Power Failure

13.3 Fire Safety SECTION 14: Appendices Appendix A- Emergency Call List

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 References:

1. HHS Publication No. (CDC) 93-8395, Biosafety in the Microbiological and Biomedical Laboratories, U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health, 5th edition, 2007, U.S. Government Printing Office, Washington DC.

2. Laboratory Safety: Principles and Practices, 2nd edition, 1995. Fleming, Richardson,

Tulis and Vesley, editors.

3. Clinical Microbiology Reviews, Oct. 1997, p. 597-610. Uses of Inorganic Hypochlorite (Bleach) in Health-Care Facilities.

4. Clinical Microbiology Reviews, Jan 1999, p. 147-179. Antiseptics and Disinfectants:

Activity, Action and Resistance.

5. Standard Operating Procedures for BSL-3 Laboratory, Dr. Tanya Popvic, Center for Disease Control and Prevention, National Center for Infectious Diseases, Atlanta, Georgia, personal communication, 01/2000.

6. Nick Combs, UNMC Facility Services. Personal communication, 2/2000.

7. UNMC/UNO/NMC Biosafety Manual, Jan 2003.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 1 Introduction 1.1 Purpose:

A. This manual is written specifically for laboratory personnel working in the DRC 8021 Biosafety Level-3 (BSL-3) laboratory. The manual is meant to be a reference guide for safe laboratory practices and procedures to be followed when working with organisms including bacteria, viruses, fungi, biotoxins, etc. that are classified as belonging to one of these categories including:

1. HIV-1 2. Risk Group-3

3. Biosafety Level-2 Containment 1.2 Scope: A. The knowledge of information necessary before working in the lab is not all-

inclusive in this manual. This manual is site specific and is meant to augment the information found in the following manuals located either in the DRC 8021 Lab area or the following websites as listed below.

1. The UNMC/UNO/NMC Biosafety Manual: www.unmc.edu/ibc/index.cfm?L1_ID=4&CONREF=8 2. The UNMC/NMC Emergency Procedure Manual: http://info.unmc.edu/safety/ 3. BSL-3 Laboratory Equipment Manual: Located in the anteroom of DRC 8021 Lab 4. BSL-3 NIH and BMBL Manuals: http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm 5. Public Health of Canadian-Health Safety sheets: www.phac-aspc.gc.ca/msds-ftss/ 6. UNMC IBC Policies: www.unmc.edu/ibc

1.3 Biosafety Training:

A. Review and understanding of the information included in this manual, and information in the manuals listed above, will be required before any laboratorian is allowed to enter and conduct research or diagnostic testing in the BSL-3 laboratory. In addition, the Principal Investigator (PI) and their staff will be thoroughly trained in BSL-3 procedures and practices by the BSL-3 Facility Manager, or designee, before work can be conducted.


The BSL-3 Facility Manager will maintain records of all laboratory training for the PI and their staff for a period of three years.

1.4. Biosecurity Clearance: A. A Security Risk Assessment for personnel working with HIV-1 or other Non-

Select Agents in the BL-3 laboratory will not be necessary.

1.5 Employee Responsibility:

A. Laboratory personnel at all levels are responsible for:

1. Complying with such UNMC/NHS Occupational Health and Safety standards, rules, regulations, and orders as are applicable to an employee's action and conduct.

2. Promptly advising their supervisor regarding all work related incidents resulting in personal injury, illness and/or property damage

3. Promptly report to their supervisor, the BSL-3 Safety and Security Manager and/or the BSL-3 Facility Manager any unsafe or unhealthful conditions in the work environment

4. Taking all necessary and appropriate safety precautions to protect themselves, other personnel and the environment. This includes the proper use of PPE while working in the BSL-3 laboratory.

5. Immediately reporting lost or stolen Proximity ID Cards to the BSL-3 Safety and Security Manager who will than have Campus Security inactivate the card to prevent unauthorized access.

Written by: Tony Sambol Date: 03/2003 Revised by: Myhanh Che 08/2009

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 2 Administration This section outlines the administrative format that is in place for the DRC Biosafety Level-3 Laboratory. Title: Personnel: 2.1 Laboratory Director Howard E. Gendelman, MD

Larson Professor of Internal Medicine and Infectious Diseases Chair, Department of Pharmacology & Experimental Neuroscience

2.2 Safety and Security Manager: Santhi Gorantla, Ph.D. Associate Professor, Department of Pharmacology & Experimental Neuroscience

2.3 Facility Manager: Myhanh Che Manager, Department of Pharmacology & Experimental Neuroscience

2.4 Biosafety Compliance Manager: Peter Iwen, Ph.D. Associate Professor,

Department of Pathology and Microbiology Biosafety Officer, UNMC/UNO/NHS


3 Administrative Duties This section defines the roles and responsibilities of each person or administrative office for the DRC BSL-3 Laboratory.

3.1 Laboratory Director

A. Position Requirements: the Biological Safety Level-3 (BSL-3) Laboratory Director shall have an M.D. or Ph.D., and have advanced training and/or experience in infectious diseases and biosafety.

B. Reports to: the Chairperson of the BSL-3 Laboratory Advisory Council C. Duties: the BSL-3 Laboratory Director shall have the authority and responsibility

to ensure that research conducted in the BSL-3 Laboratory is in compliance with the guidelines established in the Institutional Biosafety Manual and that researchers comply with the protocols approved by the Institutional Biosafety Committee (IBC). The Director shall determine the severity of all discrepancies or violations and report these to the Chair of the BSL-3 Laboratory Advisory Council. The Director shall oversee the following aspects of the laboratory including:

1. Safety, including biosafety 2. Security, including quarterly review of door access logs

3. Equipment 4. Operating budget 5. User fee collection

6. Appointment of administrative positions 7. Final access approval of Principal Investigators (PI) and staff 8. Approval of amendments to IBC approved research protocols 3.2 Safety and Security Manager

A. Position Requirements: the BSL-3 Safety and Security Manager shall have advanced training and certification in biological safety through an accrediting organization such as the American Biological Safety Association (ABSA), the

National Registry of Microbiologists (NRM) or the American Society of Clinical Pathologists (ASCP).


B. Reports to: the BSL-3 Laboratory Director.

C. Duties: the BSL-3 Safety and Security Manager duties shall include all aspects of laboratory safety and security, including biosafety and biosecurity. All discrepancies or violations shall be recorded and reported to the BSL-3 Laboratory Director. The BSL-3 Safety and Security Manager shall oversee the following aspects of the laboratory including:

1. Laboratory inspections

2. Ensuring compliance with the procedures and protocols of the Institutional Biosafety Manual

3. Responsible for content of the BSL-3 Laboratory Manual 4. Responsible for biosafety training of personnel in working with Risk Group 3 (RG-3) agents. 5. Develops and implements protocols and procedures that ensure the

security of the BSL-3 laboratory 6. Develops and implements an BSL-3 Emergency Response Plan 7. Maintain BSL-3 personnel laboratory access records 8. Inventory of BSL-3 Access Keys and I.D. cards for personnel 9. Weekly review of BL-3 entry records 10. Serves as the liaison to the UNMC Campus Security office 11. Performs routine BSL-3 ultraviolet light checks of the Biological Safety

Cabinets (BSCs) in the facility

3.3 Facility Manager

A. Position Requirements: the BSL-3 Facility Manager shall have two or more years experience in laboratory management, and shall have an in-depth understanding of BSL-3 guidelines.

B. Reports to: the BSL-3 Laboratory Director.

C. Duties: the BSL-3 Facility Manager shall oversee the following aspects of the laboratory including:

1. Scheduling routine maintenance of laboratory equipment 2. Supervision of maintenance personnel working in the BSL-3 laboratory 3. Maintaining records of BSL-3 maintenance work 4. Laboratory cleanliness 5. Stocking and ordering all “common” laboratory supplies


6. Oversees operating budget with BSL-3 Laboratory Director 7. Recording and reporting of discrepancies or violations to the Biosafety

Compliance Manager (BCM). 8. Ensuring the necessary BSL-3 laboratory “signage” is posted in

appropriate locations 9. Monitors BSL-3 autoclave performance by overseeing quality assurance

testing records for the autoclave 10. Performing weekly checks on the BSL-3 emergency lighting and eyewash

station; records results in record book.

3.4 Biosafety Compliance Manager

A. Position Requirements: the Institutional Biosafety Officer (BSO) shall fill the BSL-3 Biosafety Compliance Manager position. The BSL-3 Biosafety Compliance Manager (BCM) shall act as an independent entity that will “police” all aspects of the laboratory. All discrepancies or violations shall be recorded and reported to the Laboratory Director.

B. Reports to: the BSL-3 Laboratory Advisory Council Chair.

C. Duties: the BSL-3 Biosafety Compliance Manager shall oversee the following aspects of the BSL-3 laboratory including:

1. Annual review of the BSL-3 Laboratory Manual 2. Performing annual and unannounced biosafety inspections of the BSL-3

laboratory 3. Performs annual and unannounced audits of Select Agents 4. Maintains current inventory record of Select Agents 5. Performs annual and unannounced reviews of BSL-3 personnel access

records 3.5 Laboratory Advisory Counsel:

A. Composition: The Institutional BSL-3 Laboratory Advisory Council (LAC) is composed of the following members:

1. Institution-wide: A. LAC Chair Dr. David Crouse B. Biosafety Compliance Manager Dr. Peter Iwen C. UNMC IBC Chair Dr. Oksana Lockridge 2. Wittson Hall:

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 A. Laboratory Director Dr. Steven Hinrichs B. Safety and Security Manager Mr. Anthony Sambol C. Facility Manager Ms. Rhonda Noel D. Principal Investigators _________________ 3. Swanson Hall: A. Laboratory Director Dr. Howard Gendelman

B. Safety and Security Manager Mr. Anthony Sambol C. Facility Manager Ms. Myhanh Che D. Principal Investigators _________________

4. Durham Research Center Excellence: A. Laboratory Director Dr. Howard Gendelman

B. Safety and Security Manager Mr. Anthony Sambol C. Facility Manager Ms. Myhanh Che D. Principal Investigators __________________

B. Reports to: the Chancellor of the UNMC. C. Duties: the Laboratory Advisory Council (LAC) provides advice,

recommendations, and guidelines as to the safe operation of the BSL-3 laboratories. The LAC will provide a mechanism by which Principal Investigators (PI) and laboratorians will have a voice in the generation and management of the BSL-3 laboratories. The LAC will operate principally by emails sent to the LAC Chair, which will then be reviewed and sent out for further review and discussion to the other committee members. If necessary, a LAC meeting will be held to discuss pertinent issues relating to the laboratory operation.

Written by: Tony Sambol Date: 02/2003 Revised: Myhanh Che 08/2009


4 Biosafety Personnel Training Requirements

Pre-research blood test (only for those involved in HIV-1 research) and Biosafety Training including Biosafety exam with a passing score of 90% will be required of all personnel working with HIV-1, which is a Risk Group-3 (RG-3) agent, in the Durham Research Center 8021 BSL-3 laboratory.

4.1 Procedure

A. Proper training of the Principal Investigator (PI) and support staff performing research is required before access to the BSL-3 laboratory can be granted. All required laboratory access records will be submitted to the BSL-3 Safety and Security Manager. The manager will review the records before submission to the BSL-3 Laboratory Director for approval.

B. After fulfillment of the initial requirements listed below, the BSL-3 Laboratory Director will give written approval for the PI and support staff to begin training. Upon successful completion of this training, the manager will submit all records to the Laboratory Director for review before final approval.

4.2 Biosafety Requirements for Working with HIV-1 A. Review, successful test completion, and sign-off of the following manuals:

1. UNMC/UNO/NHS Institutional Biosafety Manual 2. UNMC/NHS Emergency Preparedness Manual 3. BL-3 Standard Operations Procedure (SOP) Manual 4. NIH-BMBL-MSDS Manual

B. Employee safety training by the UNMC/NHS Safety Department:

1. Chemical Safety (if required) 2. Radiation Safety (if required) 3. Bloodborne Pathogens

4. General Biosafety & Biosafety Level-3 5. Other training as required by IBC

4.3 Training


After the requirements for working HIV-1 have been fulfilled, the BSL-3 Safety and Security Manager will submit the personnel records to the BSL-3 Laboratory Director for approval. After approval is given, then the Safety and Security Manager will proceed with the training of the PI and any support staff to include:

1. BSL-3 Standard Operating Procedures

2. Proper usage of laboratory equipment

3. Proper security access

4. BSL-3 Biosafety principles and procedures

5. PI research specific procedures 4.4 Final Approval

Upon successful completion of training, the BSL-3 Safety and Security Manager will submit the final records to the BSL-3 Laboratory Director for review. Upon satisfactory review, final approval will be granted for laboratory access. At this time the BSL-3 Safety and Security Manager will approve the issuing of I.D. cards for the PI and staff.

4.5 Personnel Changes

Personnel changes will have to follow the laboratory access procedure to be approved by the Laboratory Director.

4.6 Annual Requirements Complete annual on-line training courses as mentioned above. Written by: Tony Sambol Date: 02/2003 Revised: Myhanh Che 08/2009

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 5 Laboratory Biosecurity As described in the UNMC/UNO Biosafety Manual, Biosecurity is defined “as protection of high-consequence microbial agents and toxins, or critical relevant information against theft or diversion by those who intend to pursue intentional misuse.” This section outlines the BSL-3 laboratory access requirements. 5.1 Personnel Requirements for Laboratory Access

Access to the BSL-3 laboratory located in Durham Research Center (DRC) 8021 is restricted at the discretion of the BSL-3 Laboratory Director. Only personnel that have completed all items on the “Biosecurity and Biosafety BSL-3 Laboratory Access Requirement Checklist” and have been approved by the DRC BSL-3 Laboratory Director will be allowed to have access to the BSL-3 laboratory. The access information will be supplied by the DRC BSL-3 Safety and Security Manager. See Section 4.

5.2 Environmental Services

Environmental Services staff will not be allowed access to the BSL-3 Laboratory.

5.3 Maintenance Personnel Access A. The BSL-3 Facility Manager will accompany maintenance workers entering the

BSL-3 laboratory. Unauthorized personnel must be accompanied at all times if working in any area of the BL-3 lab, excluding the gowning room.

B. All workers or visitors will need to read, complete and pass the “Visitor Training

for BSL-3 laboratories at the UNMC”. Completed forms will be kept by the BSL-3 Safety and Security Manager.

C. All workers entering the lab must wear double gloves, gowns and booties to enter.

No work should be done in the BSL-3 laboratory during this time. D. This information will be obtained and kept on file by the BSL-3 Facility Manager. 5.4 Security Breaches and Emergencies A. The Nebraska Medical Center Security Dispatch Center monitors the activity of

the primary entrance of the BSL-3 laboratory using a surveillance camera and a door alarm. If an unauthorized entrance into the laboratory is attempted or


occurs during this time frame, security personnel will immediately be dispatched to the laboratory to investigate, and the Safety and Security Manager will be contacted using the “Emergency Call-Down” list in the Security Dispatch Center.

B. The inner door allowing access to the biosecure hallway and the working suites is

secured by a Proximity ID card reader. This card reader is wired to the emergency electrical circuit and will allow and record entrance and egress during a time of electrical failure to the lab.

C. Laboratory access requires both the ID card and a unique code for each person

authorized to enter this area. If there were an attempt at a forced-entry, the Security Dispatch Center this would immediately notify the Security NPHL and the above- mentioned Security actions would be put into effect.

D. If evidence of an unauthorized entry and/or theft occurred, the Safety and Security

Manager will contact the Laboratory Director who will then contact the FBI and the appropriate State Health Officials.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 6 Personal Protective Equipment

6.1 Introduction

A. Personal Protective Equipment (PPE) is used to protect laboratorians from the risk of injury by creating a barrier against workplace hazards. PPE is not a substitute for good engineering or administrative controls or good work practices. It should be used in conjunction with these controls to ensure the safety and health of laboratorians. PPE will be provided, used, and maintained when working the BSL-3 laboratory. Such use will lessen the likelihood of occupational injury and/or illness. The CDC/NIH guidelines (BMBL, 4TH ed.) for biocontainment practices recommend the use of PPE including eye, face, respiratory, head, foot, and hand protection.

B. The use of Personal Protective Equipment includes:

1. Responsibilities of Principal Investigators, Support Staff, and the Safety and Security Manager

2. Institutional Biosafety Committee (IBC) biohazard assessment and PPE selection

3. Training for Principal Investigators and their staff

4. Record keeping requirements

6.2 Responsibilities

A. Principal Investigators (PI)

1. PI’s have the primary responsibility for implementation of PPE usage by their staff. This involves:

a. Providing appropriate PPE, as required in their IBC protocol, and making it available to their support staff

b. Ensuring support staff has received the training on the proper use, care, and cleaning of PPE.


c. Maintaining records on PPE assignments and training

d. Supervising employees properly use and care for PPE.

e. Seeking assistance from Safety and Security Manager to evaluate biohazards

f. Notifying the Safety and Security Manager when new hazards are introduced or when processes are added or changed.

g. Ensuring defective or damaged equipment is immediately replaced and/or reported to the Facility Manager.

B. Principal Investigator’s Support Staff:

1. The Support Staff are responsible for following:

a. Wearing PPE as required in IBC protocol

b. Attending required training sessions.

c. Caring for, cleaning, and maintaining PPE as required

d. Informing the Safety and Security Manager of the need to repair and/or replace PPE.

C. Safety and Security Manager

1. The Safety and Security Manager is responsible for the training and proper usage of the PPE. This involves:

a. Conducting workplace assessments to determine the presence of biohazards that necessitate the use of PPE

b. Conducting periodic workplace reassessments as requested by supervisors and/or as determined by Employee Health

c. Maintaining records on biohazard assessments

d. Providing training and technical assistance to Principal Investigators and support staff on the proper use, care, and cleaning of IBC required PPE.


e. Providing guidance to the Principal Investigator for the selection and use of approved PPE as required by the IBC

f. Periodically reevaluating the suitability of previously selected PPE

g. Reviewing, updating, and evaluating the overall effectiveness of the PPE training and use

6.3 Selection and Use of Personal Protective Equipment

PPE will be required to reduce the risk of exposure of all laboratorians by contact, inhalation or ingestion of an infectious agent, or toxic substance. For biological agents, the IBC, with input from the Principal Investigator, will determine the appropriate Biosafety Level under which the research will be conducted. Work in the BSL-3 lab will require the appropriate type of PPE be worn.

1. Laboratory Coats and Gowns

Coveralls will be used to protect street clothing against biological or chemical spills as well as to provide some additional body protection. These will be changed out on an “as needed” basis or minimally monthly. Lab coats will be required for corridor, equipment room/suite B and autoclave room.

2. Foot Protection

For general biological lab use, comfortable shoes such as tennis shoes or nurses shoes can be worn. Sandals and other types of open-toed shoes are not permitted in the BSL-3 lab due to the potential exposure to infectious agents or toxic materials as well as physical injuries associated with the work. Booties will be required at all times for all lab locations.

3. Face shields and Eye Protection

Face shields and goggles should be worn whenever procedures with a high potential for creating aerosols are conducted. These include harvesting of tissue culture fluids and manipulations of high concentrations or large volumes of infectious materials.

4. Gloves


“Double-Gloving” is required at all times in the BSL-3 laboratory when working with infectious or toxic agents.

5. N-95 Respirators

All personal working with Risk-Group 3 (RG-3) organisms, or as directly by the approved IBC protocol are required to be “fit-tested” by Employee Health to determine what type and size of N-95 respirator mask needs to be worn. An annual N-95 fit test is required.

6. Powdered Air Purifying Respirators (PAPRs) See section on PAPRs in the Laboratory Equipment Manual.

Note: Use of PAPRs is optional and only needed to be used if required by the Institutional Biosafety Committee for an individual PI’s research protocol.

A. Personnel will be required to have a physical, including a spirometry test, conducted by Employee Health before being allowed access to PAPRs units.

B. The Safety and Security Manager will train personnel on the use of PAPRs, i.e. what size they should wear, proper way to don equipment, checking air flow rates, and battery life. Training will be documented.

C. It is important that all PAPRs be kept clean and properly maintained. Cleaning is particularly important for eye and face protection where dirty or fogged lenses could impair vision. PAPRs should be inspected, cleaned, and maintained at regular intervals so that the PAPR provides the requisite protection. PPE, including PAPRs shall not be shared between employees. PAPRs will be distributed for individual use.

D. All single-use PPE ( gloves, N-95 masks)shall be disposed of daily by placing into the appropriate biohazard bins located in the egress area.

6.4 Training

A. All laboratorians working in the BSL-3 laboratory shall receive training in the proper use and care of PPE. Annual, or as needed, refresher training shall be offered by the BSL-3 Safety and Security Manager to both the Principal Investigator and support staff, as needed. The training shall include, but not necessarily be limited to, the following subjects:

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 1. Why PPE is necessary to be worn 2. What PPE is necessary 3. How to properly don, doff, adjust, and wear PPE 4. The limitations of the PPE 5. The proper care, maintenance, useful life and disposal of the PPE

B. After the training, the employees shall demonstrate that they understand how to use PPE properly, or they shall be retrained before working in the lab will be allowed.

C. N-95 fit testing is required annually.

6.5 Recordkeeping

Written records shall be kept of the names of persons trained, the type of training provided, and the dates when training occurred. The BSL-3 Safety and Security manager shall maintain employees’ training records. The Safety and Security Manager shall maintain the approved Institutional Biosafety Committee protocol for each approved research project.


7 Laboratory Signs and Tags

Signs and tags are used to indicate hazards that may exist in the laboratory. All laboratorians utilizing the Durham Research Center (DRC) BSL-3 facility must be aware of the proper use of warning signs, tags and symbols and the hazards they represent.

7.1 Proper use of Signs and Tags:

A. Laboratory signage will be posted on the outer door of the BSL-3 laboratory as required. (See below) Signs and tags are not intended as substitutes for preferred abatement methods such as engineering controls, substitution, isolation, or safe work practices. Rather, they are additional safety guidance and increase the laboratorians’ awareness of potentially hazardous situations.

B. Tags are temporary means of warning all concerned of hazardous conditions, defective equipment, etc. Tags are not to be considered as a complete warning method, but should only be used until a positive means can be employed to eliminate the hazard; for example, a "Do Not Start" tag is affixed to a machine and is used only until the machine can be locked out, de-energized, or inactivated.

7.2 Posting of Signs and Tags

A. Any laboratorian that becomes aware of an unsafe condition will immediately advise the Safety and Security Manager and/or the Facility Manager of that condition. Either manager will determine whether a tag or sign is needed and, if so, that the appropriate sign or tag is posted or attached as required. If either manager is not available, the laboratorian will phone the Institutional Biosafety Officer (BSO) and request assistance.

B. Either manager will evaluate the situation and initiate appropriate corrective action. Either manager is responsible for removing the sign or tag only after the unsafe condition has been corrected.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 7.3 Hazard Warning Signs and Labels

Hazard Categories: Hazard identification signage has four distinct categories:

1. NOTICE – states a policy related to safety of personnel or protection of property but are not for use with a physical hazard.

2. CAUTION – indicates a potentially hazardous situation that, if not avoided, may

result in minor or moderate injury.

3. WARNING – indicates a potentially hazardous situation that, if not avoided, will result in death or serious injury.

4. DANGER – indicates an imminently hazardous situation that, if not avoided, will

result in death or serious injury.

7.4 Examples of Signs:

The following are examples of signs/notices that may be posted by the door entering into the Biosafety Level-3 (BSL-3) Laboratory, or on doors or equipment located inside the facility. Each sign/notice should be marked with a time/date, action taken and person leaving the notice.

1. Notice signs: No Unauthorized Personnel Admitted

2. Biological Hazard Signs: The following is an excerpt from the O.H.S.A. Federal Register:


“Biological hazard signs: The biological hazard warning shall be used to signify the actual or potential presence of a biohazard and to identify equipment, containers, rooms, materials, experimental animals or combinations thereof, which contains, or are contaminated with, viable hazardous agents. For the purpose of this subparagraph the term “biological hazard,” or “biohazard,” shall include only those infectious agents presenting a risk or potential risk to the well being of man. The biohazard symbol shall be designed and proportioned ……”

3. Caution Signs: "Caution" signs shall be used to warn of a potential hazard or to

caution against unsafe practices, and to prescribe the precaution that will be taken to protect personnel and property from mishap probability. The sign shall be of yellow and black colors.

A. “Biological Safety Cabinet not working.” B. “Autoclave not working” C. “Infectious Material in 8021___, to be decontaminated” D. “Chemical Spill has occurred- Do Not Enter”

4. Warning/Danger Signs: “Warning or Danger" signs shall be used where an immediate hazard exists and specific precautions are required to protect personnel or property. The sign shall be of red, black, and white colors. These signs are found in the “Signs” folder in the BSL-3 anteroom shelf. Some examples are:

. A. “Biological Spill has occurred- Do Not Enter” B. “BSC Ultraviolet lamps not working in 8021___” C. “Exhaust System Failure-Do Not Enter” D. “Security breach detected- laboratory access denied” E. “BSC Malfunction”


8 Workflow: Laboratory Entrance and Egress Critical to laboratory biosafety is the proper methods used to enter and exit the

laboratory. Improper practices used for laboratory entrance or egress will compromise the secondary barriers, i.e. facility design and operational practices, that were meant to protect the environment external to the laboratory. A thorough understanding of the Durham Research Center (DRC) BSL-3 laboratory design is necessary to ensure secondary barrier protection is not compromised.

8.1 Entering the Lab A. Approved personnel enter DRC 8021 anteroom using swipe card. All rooms of

the Biosafety Level-3 (BL-3) laboratory, excluding the first room, which is the changing or anteroom, will be considered “dirty.” Full Personal Protective Equipment (PPE) will be required for anyone to enter into the lab past this changing room.

Access into the Biosecure Corridor and all interior rooms including the working

suites requires the pre-approved coding of your UNMC I.D. proximity access card and personal access number into the proximity-card keypad mechanism. This security access is given only by the Safety and Security Manager, after approval by the Laboratory Director, and is programmed into the system by the personnel in the Security Department. All doors must be kept closed and locked at all times. Doors are propped open for 45 seconds or longer will set off the alarm.

B. While in the anteroom, remove any lab coats or jackets worn up to this point. Put

in pass-through locker and will be picked up from the exiting room. Personal protective equipment (PPE) including booties, gowns (if none already in-use), N-95 respirator masks and double gloves must be put on. Hairnets and hospital scrubs are optional. Note: Eye protection is required if the possibility of splashing will occur or if contacts as worn. Personnel are then ready to work in the BSL-3 laboratory.

C. If visitors or maintenance personnel are entering, record laboratory entrance

information on the “BSL-3 Area Entry Log” that is kept in the anteroom.


D. Before entering the laboratory, inspect the visual monitoring device that indicates and confirms that the one-pass directional inward (negative) airflow is occurring. DO NOT ENTER the lab if a red light is on or audible alarm is on. Post the “Exhaust System Failure” sign on the door

and contact the Facilities Manager and/or the Safety and Security Manager. (See Sections 7 and 9)

E. Make sure that the Proximity Access Card is under your PPE before entering the

lab. F. Two sterilizers in autoclave room are pass through units

with the dirty side in 8021 H and clean side in 8017. 8.2 Working at the Biological Safety Cabinet (BSC) Inside Each Suite

A. Needles, scalpels or other sharps are discouraged in the HIV area; they can be used only when absolutely necessary. They must be discarded carefully in the red sharps containers.

B. Avoid working with glass in the HIV area; permissible items include: hemocytometers, slides, coverslips, small bottles in kits, etc. Use extreme caution when working with glass items. All glass bottles will be stored in a dedicated cabinet, not on countertops or the top of refrigerators. Empty (reusable) bottles are cleaned with ethanol and removed from the BSL-3.

C. Universal precautions must be used when working with any human tissue or blood products, including media made with human sera. Treat all blood or tissue products as though they are contaminated with HIV, hepatitis, etc.

D. HIV-infected material can only be vortexed in the BSC. Any manipulation with HIV-infected material must be done in the BSC.

E. Squirt bottles with bleach and 70% ethanol should always be present in hood when working with HIV.

F. Any spill or droplets inside the BSC should be cleaned immediately by placing an absorbent towel over the spill and immediately wetted with 70% ethanol before continuing work.

G. A bucket filled with approximately 1 inch of 50:50 bleach and water must be present in the hood for all work.

H. You should change your outer pair of gloves after working with 10X HIV-1, or following any contamination of your gloves.

I. Pipette tips, cryovials, eppendorf tubes, and 6ml snap-cap tubes can be discarded

in the bleach buckets. Make sure tips and tubes are empty prior to discarding, or they will not get bleached properly. Prior to removing buckets, make sure all


tubes, tips, etc are completely submerged in bleach and no pink media is visible. Add more bleach if necessary.

J. Pipettes should be bleached after use and drained of bleach prior the disposal in the biohazard trash.

K. All other reagents or supplies used in the BSC should be removed when your work is completed. This includes tips, bleach buckets, etc.

L. Before disposal of culture flasks and larger tubes (12ml snap-caps, 15ml or larger conical), squirt a small amount of bleach into tube, cap and swirl. Empty the bleach into bleach bucket, lightly cap flask or tube, and place in the biohazard trash.

M. Teflon flasks are not discarded. Add some bleach, cap, swirl and leave in hood until you are done working. Then, empty the bleach into the bucket, remove the flasks to the sink and fill with 1/3 bleach and then to the top with water. Leave them to sit in collection container on the cart next to sink overnight, then empty, rinse, and clean with ethanol, autoclave and remove from the BSL-3.

N. Vacuum traps should be emptied after use (and before full and overflowing), and filled with 1 inch of 50:50 bleach water mixture.

O. All equipment in the hood must remain inside hood, i.e., electronic pipetter, traps and tubing. Only when necessary (i.e., for repairs or cleaning), should equipment be removed. The equipment should then first be disinfected with 70% ethanol.

P. Equipment present in the HIV lab must remain in the HIV lab, unless permission is obtained from the facility manager. Equipment must be cleaned with ethanol prior to removal.

Q. Plates and other tubes, etc. can only be brought out of the BSL-3 if HIV-1 has been inactivated (by RT assay procedures, MTT reagents, ELISA detergents, Trizol extraction, etc.). All containers must be wiped with ethanol prior to leaving P3.

R. See Radiation Safety Rules for how to handle radiation in the BSL-3. 8.3 Maintenance and Cleaning

A. Biohazard waste bags should never be filled more than 75% full! B. All BSL-3 trash must be double-bagged, and must be autoclaved. C. The autoclave trash should not contain media or other liquids. Media must be

bleached. When autoclave trash is carried out to autoclave room, bags must be in a leak proof container.

D. Empty pipette tip boxes should have tape removed, and be placed into a biohazard bag in the BSL-3 (Double bag as usual). When full, remove bag, autoclave, and do not discard. Mark “tip boxes” on the bag. These will be packed with new tips, and reused.

E. Pick up loose pipettes, tubes, etc. on the floor, as these can present a slip hazard.


F. On occasion, maintenance men may have to enter the BSL-3. They must be let in by the Facility Manager or a designated person (do not give out the key pad code), and must double-glove and gown to enter. No work should be done in the BSL-3 at this time.

8.4 Exiting the Lab

A. After work is completed in the BSC, decontaminate all work surfaces. All contaminated materials must be cleaned up inside the BSC and placed into the biohazard bags. Outer pair of gloves are to be discarded in biohazard bag before leaving the suite.

B. The BSC should be left running at all times. If the UV light is used to decontaminate the exposed surfaces of the BSC, lower the sash, turn the UV light timer for 60 minutes then press on the BSC UV light. Note: The BSC UV light will only come “on” when the sash is lowered completely to the sash resisting stops.

C. To autoclave, follow instruction posted next to autoclaves. For each cycle,

a chemical indicator or steam indicator strip, and autoclave tape will be placed into the autoclave.

D. The PPE should be removed in this order: hairnet, gown (see below),

booties and mask. The (inner) gloves will always be removed last. All PPE will be placed into one of two appropriately labeled” hands-free” biohazard bins containing autoclavable biohazard bags. These contents of the bins will be autoclaved when either ¾ full or weekly.

E. Before leaving the autoclave room and exiting the laboratory, hands must

be washed thoroughly using an antiseptic soap and dried.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 9 Laboratory Equipment

Laboratorians using the BL-3 equipment contained in the common equipment room or either of the working suites must be knowledgeable in the proper use of the equipment to ensure not only their safety from mechanical dangers, but biosafety from biological agents that they may be manipulating in their work.

9.1 General Notes:

A. All equipment present or brought into the BL-3 laboratory must remain in the lab unless permission is obtained from the Facilities Manager to remove it.

B. If there is a malfunction with any of the laboratory equipment, notify the Facilities

Manager. 9.2 Equipment Certification and Safety Checks:

A. The BSC and the laboratory ventilation system (HVAC) will be certified on an annual basis. The Facilities Manager keeps these records. Refer to the Institutional Biosafety Manual for further information.

B All electrical equipment will be inspected and certified before bringing into the laboratory by the UNMC Electrical Safety Department. Equipment will be checked on a regular basis after that. The Electrical Safety Department will keep records

9.3 Gas Canisters:

A. Canisters should all be restrained and marked with the tag to indicate if they are “Full”, “In use”, or “Empty.”

B. All canisters not being used should have the canister cap secured onto the top of the tank. D. Notify the Facilities Manager when a tank becomes empty so that it can be replaced before all the tanks are empty.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 9.4 Laboratory Exhaust System:

A. Single-pass ventilation is supplied to all laboratory areas. Doors to laboratories must be kept closed, as containment of hazardous materials is partially dependent on proper balance of airflow. Disruption of the positive pressure in the corridor by a laboratory door opened for an extended period may result in transmission of airborne materials from the laboratory to the corridor.

B. Before beginning work, visually verify that the negative airflow system for the laboratory is functioning correctly by observing the monitor just right outside of each room. When Exhaust System fail, audible alarm will sound -Do Not Enter. Call Facility Manager 350-3799.

9.5 Biological Safety Cabinets: A. Before beginning work in the BSC, visually verify that the BSC should be turned

“on” (if not already running), and the magna-helical gauge should read > 0.4 inches. Turn on the BSC for a minimum of 15 minutes prior to use. If either indicator appears incorrect, do not proceed with any laboratory work. Contact the Facilities Manager to have the system inspected and problem corrected.

Biological Safety Cabinets (BSC) Properly maintained BSCs, when used in conjunction with good microbiological techniques, provide an effective containment system for safe manipulation of moderate and high-risk microorganisms (Biosafety Level 2 and 3 agents). Both Class I and II BSCs have inward face velocities (75-100 linear feet per minute) that provide comparable levels of containment to protect laboratory workers and the immediate environment from infectious aerosols generated within the cabinet. Class II BSCs also protect the research material itself through high-efficiency particulate air filtration (HEPA filtration) of the airflow down across the work surface (vertical laminar flow). Class III cabinets offer the maximum protection to laboratory personnel, the community, and the environment because all hazardous materials are contained in a totally enclosed, H – high E – efficiency

ventilated cabinet. P – particulate A – air

9.6 Centrifuges Procedure

A. Check centrifuge tubes for cracks/chips before use. B. Do not fill centrifuge tubes to the very top of the tube. C. Tightly seal all centrifuge tubes or use safety cups/ buckets to prevent aerosol escape.


D. Use a Biological Safety Cabinet (BSC) to load and open tubes, safety cups, and buckets when working with biohazardous materials. Decontaminate tubes, safety cups, and buckets before removal from the BSC and transport to the centrifuge.

E. Allow the centrifuge to come to a complete stop before opening.

9.7 Ultra-Centrifuges (In addition to the above): A. Clean rotors, lids, adapters, and associated parts with 70% EtOH.

B. Make sure that rotors are locked to the spindle and that buckets are properly seated on their pins. Only use the rotor handle tool to tighten ultra speed lids.

C. Do not use rotors that have been dropped or struck against a hard surface. D. Contact your centrifuge representative for specific information. 9.8 Ultra-low Freezer

The ultralow freezer, in the common equipment room, is wired to a remote temperature alarm. If the temperature falls below the programmed set point, the alarm will be activated and the Facility Manager will be notified.

9.9 Equipment decontamination: A. All contaminated equipment located in the BL-3 laboratory will be decontaminated using a 0.5% Sodium hypochlorite (10% bleach) solution that is made at the beginning of each working day and follow by 70% ethanol. The decontaminated equipment will be required to stand for 30 minutes before removal from the laboratory for repair, maintenance or packaging for transport.

B. Laboratory equipment and work surfaces will be decontaminated routinely with a 0.5% Sodium hypochlorite (10% bleach) solution follow by 70% ethanol after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials.

C. The BSC will be left running after they are done being used. The UV lamps will

be turned “on” for a minimum of one hour in the BSCs 9.10 Biological Safety Cabinet Decontamination

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 A. Gaseous decontamination of High Energy Particulate Air-filters (HEPA) in the BSC is required whenever the BSC is moved or the HEPA needs to be replaced. Decontamination is usually carried out with formaldehyde sublimed by heat from Para- formaldehyde flakes in the presence of heat and high humidity. The UNMC/NHS contracts with BALCON for these services. 9.11 Laboratory Facility Decontamination A. Decontamination of a laboratory facility or even a single contained room is carried out by sealing off the facility or room and performing gaseous decontamination in the same fashion used for BSC. The UNMC/NHS contracts with BALCON for these services. University policy requires posted notification at least one week prior to this occurring.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 10 Working in a Biological Safety Cabinet

A Biological Safety Cabinet (BSC) is a type of primary barrier used to contain infectious material that the laboratorian is working with. In the BL-3 laboratory, both Class II Type “A” and “B” BSCs are used. Type “A” BSCs are free-standing and after High Efficiency Particulate Air (HEPA) filtration recirculate 30 % of the air back into the room. Type “B” BSCs are hard-ducted and vented directly to the outside of the DRC building. Proper use of, and work in, the BSC is imperative for the safety of the laboratorian.

10.1 Prior to Beginning Work

A. Laboratory personnel will receive appropriate training, by the Safety and Security

Manager, on the potential hazards associated with the work involved. Annual training will be required of all personnel. Laboratorians will receive updates and training as necessary for procedural changes.

B. Laboratory personnel will receive the appropriate training on the proper use of a Biological Safety Cabinet (BSC). An equipment manual for the BSC is located in each working suite and in the main BL-3 equipment manual in the autoclave room. C. All manipulations of infectious materials or cultures are conducted in a Class II Biosafety Cabinet (BSC). This includes sonication, preparing specimens

for -70 O C, etc. The BSC should be turned “on”, and air circulation allowed for fifteen minutes before starting work in the BSC.

D. The BSC alarm switch should be turned “on” and the protective sash should be the proper height, indicated by the yellow tape, about before starting work in the BSC.

E. Check the BSC airflow (magnehelic) gauge. It should read > 0.4 inches of water. Do not use the BSC if the airflow gauge indicates < 0.4 inches of water. Contact the Facility Manager to have the BSC inspected and repaired to correct the problem. F. If there is a drop in airflow while the BSC is in use, an alarm will sound. Immediately stop work, exit the room, and place a “DO NOT ENTER” tape/sign on the door. Notify the Facility Manager and/or the Safety and Security Manager.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 10.2 While working in the BSC the following procedures should be observed:

A. When entering and exiting the BSC, move arms in a slow, deliberate manner. Bring arms straight into and out of the BSC and avoid side-to- side motions that could disrupt airflow.

B. Do not place large pieces of equipment in the BSC. Place small equipment and supplies to the sides of the BSC. Avoid placing equipment directly in front of the work area. After material are placed in the BSC, allow 2-3 minutes before beginning work to rid the area of all “loose” contaminants that may be introduced by these items.

C. Do not place any objects on the intake and exhaust grills in the front or

back of the BSC. Place shallow discard pans/containers to the side of the BSC.

D. A plastic backed absorbent under-pad may be placed on the BSC surface

when performing activities with a high probability of droplet production or spills. 10% bleach may be poured onto the pad to increase protection.

E. All work should be done in the deepest part of the BSC for optimal

protection. Work should also proceed from “clean” to “dirty” to avoid cross-contamination. Open all containers with infectious material only within the BSC. Procedures involving agitation or sonication of cultures should be performed only within the BSC. When a procedure such as centrifugation cannot be conducted within a BSC, the appropriate combination of PPE, including N95 respirators and/or face shields, shall be worn. Vesicle containment devices such as centrifuge safety-cups or sealed rotors will be used. Centrifuge safety cups and/or sealed centrifuge rotors will only be loaded and unloaded in the BSC. (See Section 9.6/7)

10.3 Upon completion of work: A. All contaminated material should be discarded within the BSC. Do not

bring contaminated material outside the BSC without being contained in a sealed biohazard container that is then placed into a tub for decontamination. Specimens, equipment etc. that will be brought outside of the working suites or the BL-3 laboratory itself need to be wiped down with a 10 % solution of bleach if working with select agent and 70% EtOH

if working with HIV.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 B. Disinfect BSC by wiping down 70% EtOH. Discard paper towel into biohazard

bag. Leave BSC UV light on for one (1) hour. and leave the BSC running. Note: lowering sash before turning “on” UV lights.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 11 Decontamination of Biohazard Waste A. The primary responsibility for the safe handling and disposal of infectious

waste resides with the generator of the waste. This responsibility extends to the ultimate point of disposal even when there are other parties involved in the handling of the waste. Disposal or decontamination of biohazardous waste is achieved in the BL-3 laboratory largely through the use of the pass-through autoclave for solid materials, bleach at a 10% final concentration for liquids.

11.1 Standard Procedures

A. Before working inside the Biological Safety Cabinet (BSC), a bucket containing 50:50 bleach/water should be placed inside the hood for decontamination of any biohazardous liquid and all solid biohazardous materials and other infectious waste will be placed into double biohazard bags. These bags are then placed into a leak proof container (metal or plastic tub) before being brought out of the working suites. If Personal protective equipment (PPE), mainly gloves, gowns and booties, have become contaminated by a major spill these should be removed and placed into a biohazard bags. Extra booties and gowns are located in each working suite.

B. Biohazardous waste bags are to be removed, autoclaved and replaced with new

bags when 70% full.

C. Materials to be autoclaved should not contain large amounts of contaminated liquid media or other liquids; liquids must first be decontaminated by adding bleach to a final concentration of 10%, put into bleach bucket and be dumped down the sink located in the corridor.

D. Materials will be decontaminated using the “pass-through’ or “double- door” autoclave located in the BL-3 laboratory autoclave room.

E. Place a piece of autoclave tape on biohazard bag that is being autoclaved, and chemical indicator strip or a biological indicator in the autoclave towards the opposite door for each cycle.

F. For solid wastes, decontamination is performed by using the autoclave on Cycle#3, which is set for one hour at 121C, 15 psi. For liquid wastes, the

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 autoclave will be run on Cycle #4, which is set for 20 minutes at 121C, at 15psi. G. Material to be decontaminated is not to left in the autoclave either run or un-run

overnight or over the weekend. All autoclave runs should be started before 2 PM and the washroom crew notified. If you start an autoclave run after 2 PM, you are responsible for removing the decontaminated material from the other side after the run is completed.

H. All materials brought into the lab must be decontaminated before leaving the

facility. Materials removed from the lab to be use in the remainder of the scientific process, including lab instruments, must be decontaminated by wiping down with 70% EtOH. Do not bring large carriers into the lab, as they will be difficult to decontaminate before removal. Do not bring cardboard boxes into the lab- unload the contents and use a cart to bring the contents in. Paper materials including note, procedures, etc. should be UV irradiated for one hour before removal from the lab.

I. Empty plastic pipette-tip boxes must be autoclaved as above before reuse. 11.2 Autoclave Performance Monitoring A. The Facility Manager will also monitor autoclave performance weekly by using a biological indicator. A biological indicator is a self-contained, biological system that permits activation and culturing of Bacillus stearothermophilus and Bacillus subtilis (globigii) when the sterilization process is inadequate. For each run, the autoclave printout and the chemical or biological indicator strip will be logged into the autoclave performance book. All autoclave performance records will be kept by the Washroom staff and reviewed weekly by the Facility Manager. B. The Facility Manager will be notified in the event of any autoclave malfunction or incomplete autoclave run. The Facility Manager will schedule all repair and or maintainance work.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 12 Spill Clean-up Procedure A. Minor and major spills and accidents that result in overt potential

exposure, i.e. occurring outside of the Biological Safety Cabinet (BSC), to infectious materials are immediately contained and then reported to the Safety and Security Manager and the Biosafety Compliance Officer. Employee Health will provide all appropriate medical evaluation, surveillance, and treatment to all personnel present in the laboratory at the time the exposure occurred. Written records on any such instances will be maintained by Employee Health and the Safety and Security Manager.

12.1 Spills of HIV

A. Spill: should be cleaned up by placing a paper towel or other absorbent material over the spill. Add 10% bleach to soak up the spill by starting slowly at the perimeter of the spill and work inward. Allow to soak for 30 minutes after which wipe up the spill. Use forceps, towels and/or a dustpan to scoop up any broken glass placing broken glass into a “sharps” container. Decontaminate all materials as per autoclaving procedure. If your Personal Protective Equipment (PPE) has been contaminated, remove soiled PPE and place into biohazard bag. Clean PPE can be found in the cupboards in each of the working suites. Do not remove mask at this time.

B. Injury: spills of HIV with cut or puncture injuries need to be handled differently.

Contact Facility Manager 350-3799. Let the wound bleed, then wash with soap and care as other wound. Next report to the Safety and Security Manager and the Biosafety Compliance Officer. Then go to Employee Health to get appropriate medical evaluation

12.2 Personal Protective Equipment

A. If infectious material is spilled onto Personal Protective Equipment (PPE), immediately remove the lab gown and or booties, place them into an autoclave biohazard bags and replace with a clean gown and or booties located in the cupboards in the working suites. If personnel clothing becomes contaminated it should quickly be removed and autoclaved. There is shower for use and surgeon cloth that can be used temporarily. Notify

the Safety and Security Manager and Biosafety Compliance Officer and then proceed to Employee Health and fill out an incident report.

12.3 Eye and Skin Involvement


A. If HIV is spilled onto skin, the contaminated skin should be washed thoroughly with soap and water. If the skin is broken (needle stick), bleeding should be encouraged and the injury washed with copious amounts of water. (Do not use bleach on an open wound)

B. For splashes in the eye, the eye should be flushed out with copious amounts of water immediately at the eye station. Avoid rubbing the eyes. C. Notify the Safety and Security Manager and the Biosafety Compliance Officer of the incident and fill out an incident report. Inform the Biosafety Compliance Officer to have him notify Employee Health of the organism(s) that you were exposed to.

12.4 Chemical Spills

A. A chemical spill kit is located in the equipment room to be used in cases of chemical spills. Refer to the UNMC/NHS Emergency Procedure Manual for instructions on what to do and who to call in case of a chemical spills. An emergency eyewash station is located in the corridor.


13 Emergency Procedures

A. All UNMC/NHS emergency phone numbers, contact information and procedures for emergencies are located in the yellow-colored UNMC/NHS Emergency Procedure Manual located in the equipment room (Suite B). Refer to this manual for specific instructions.

13.1 Phone

A. There is a phone located in the corridor (9-2776) and a call-list of people to immediately contact about an emergency in the BL-3 lab.

13.2 Power Failure A. In the event of an electrical power outage, the emergency lights that are

located in the working suites and the autoclave room will be automatically activated. The “SmartUPS” back up power supply units for the Biological Safety Cabinets (BSC) in the working suites will automatically activate and provided up to three minutes of power for the BSC. Turn off any equipment in the BSC to allow a longer “running” time for the back-up power supply.

B. Working quickly, close down all open containers, etc. that may contain infectious materials.

C. Place any instruments, pipettes, etc. into biohazard containers. D. Gently wipe down all surfaces in the BSC using 10% bleach.

E. Exit the working suites following standard procedures.

F. In the autoclave room, discard all personal protective equipment (PPE) into the biohazard bins containing the double autoclave bags except for the N95 respirator. Wash and dry hands thoroughly and completely before exiting the laboratory. G. Remove N95 respirator after leaving the autoclave room and before

leaving the changing room.


H. Place “Exhaust System Failure- Do Not Enter” warning sign on the outer door. Restrict access until a one-hour period has elapsed, after power has been restored, that will allow for one complete change in room air and the one-hour UV decontamination time period. NOTE: The exhaust fans to the working suites will automatically come “on” upon restoration of power. The input air into the rooms will not come “on” until several minutes after that. The working suites’ air will be completely exhausted outward during this time-period. If the UV lamps don’t come “on” automatically after power restoration, wait for one hour before entering into the BL-3 lab with full PPE to activate the UV lamps.

I. Report incident to Safety and Security Manager and Facility Manager.

Appropriate medical evaluation, surveillance, and treatment will be provided. Written records on any such instances will be maintained.

13.3 Fire Safety

A. If a fire occurs in either of the working suites, notify Security by dialing 9-5111 using the phone in the corridor. Try to extinguish it if the fire is small and deemed manageable.

B. If a laboratorian is on fire, use the fire-blanket to smother a fire occurring

on the individual, and then use the Halon-gas fire extinguisher and try to extinguish the fire.

C. Exit the working suites as quickly as possible immediately after using the

fire extinguisher as Halon-gas displaces the oxygen present in the room. D. Immediately, contact Security using the number provided in the

UNMC/NHS Emergency Manual. Notify the Safety and Security Officer and the Facility Manager of the incident and fill out an incident report. Appropriate medical evaluation, surveillance, and treatment will be provided. Written records on any such instances will be maintained.

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 14 Appendix A: Emergency Call List Biosafety Level-3 Laboratory DRC Room 8021 1. Laboratory Director Dr. Howard Gendelman Office: 9-8920 Home: 330-4029 Cell: 203-5676 2. Safety and Security Manager Dr. Santhi Gorantla Office: 9-8754 Cell: 682-2436 3. Biosafety Compliance Officer Dr. Pete Iwen Office: 9-7774 Page: 888-3504 Home: 558-5314 4. Facilities Manager Ms. Myhanh Che Office: 9-5981 Home: 934-0003 Cell: 350-3799 5. Glassware/Washroom Mr. Na Ly Office: 9-3792 Autoclave Home: 502-4433 Cell: 402-881-7688 Security 9-5111 Emergency/Injury 9-5555 P-3 LAB 9-2776 UneCard Office: 9-2917 Page: 888-3735

Cell: 943-9502 Brett Cox- Balcon (BSCs/HVAC) 978-0716

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Biosafety Level 3 Laboratory Durham Research Center 8021 Laboratory Director Howard E. Gendelman, MD

Safety and Security Manager: Santhi Gorantla, Ph.D Biosafety Compliance Manager: Peter Iwen, Ph.D Facility Manager: Myhanh Che Laboratory Maintenance: Na Ly Emergency Contact: Dr. Gendelman x 9-8920 Dr. Santhi Gorantla x 9-8754 Dr. Pete Iwen x9-7774 Myhanh Che x9-5981

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